Green synthesis of chlorella-derived carbon dots and their fluorescence imaging in zebrafish

Recently, carbon dots (CDs) have been shown to exhibit exceptional water solubility, low toxicity, favorable biocompatibility, stable fluorescence properties with a wide and continuous excitation spectrum, and an adjustable emission spectrum. Their remarkable characteristics make them highly promising for applications in the field of bioimaging. Zebrafish is currently extensively studied because of its high genetic homology with humans and the applicability of disease research findings from zebrafish to humans. Therefore, spirulina, a commonly used feed additive in aquaculture, was chosen as the raw material for synthesizing fluorescent CDs using a hydrothermal method. On the one hand, CDs can modulate dopamine receptors in the brain of zebrafish, leading to an increase in dopamine production and subsequently promoting their locomotor activity. On the other hand, CDs have been shown to enhance the intestinal anti-inflammatory capacity of zebrafish. This study aimed to explore the chronic toxicity and genotoxicity of CDs in zebrafish while providing valuable insights for their future application in biological and medical fields.


Introduction
Carbon dots (CDs) have the advantages of adjustable photoluminescence, a large two-photon absorption cross section, easy functionalization, low toxicity, good chemical inertness, good dispersibility, and biocompatibility. 1,2When used in biomedicine, CDs not only can be used as a luminous indicator but also have high antibacterial efficiency. 35][6][7][8] The marine environment provides numerous resources, including plants, animals, and microorganisms, which can be used to extract polysaccharides, such as alginate, carrageenan, chitin, chitosan, agarose, ulva, and porphyrin. 9hese polysaccharides found in marine environments can serve as carbon-rich precursors for the synthesis of CDs. 10 Using marine polysaccharides to produce CDs can transform renewable energy into cutting-edge technology products.At present, there is a concern that nanomaterials may cause intergenerational effects; therefore, the toxicity of nanomaterials to organisms has become a critical consideration before the application of nanomaterials. 11lorella, as a common health care product, 12,13 also has anti-oxidation and anti-aging properties.It is oen used as a dietary supplement in aquaculture 14 since the addition of chlorella to sh diets improves enteritis caused by sh meal diets and improves sh performance against pathogens. 15At present, there are occasional reports about using chlorella to make bio-based CDs, which are oen used for detection metal ions.CDs made by chlorella through a hydrothermal method were used to detect Fe 3+ in wastewater, 16 and it was found that they could be successfully doped without additional reagents, such as nitrogen, sulfur, phosphorus, or potassium.In addition, chlorella-derived CDs can also be used to distinguish the life and death of chlorella. 17Using chlorella-derived CDs to modify ZnO can be used for photocatalytic behavior in the degradation of carbamazepine. 18ebrash is a freshwater sh that has a small size, good vitality and reproductive ability, is easy to raise and manage, 19 and has a short life cycle, going from fertilized eggs to adults in about three months, 20 meaning they can be quickly raised in large-scale experiments.In addition, zebrash larvae are transparent, so its internal structures and organ development process can be observed directly through the microscope.The use of zebrash for toxicity assessment of CDs has the following advantages: zebrash genome is 87% similar to the human genome; pathological states of many diseases and genes associated with disease causes are highly conserved among humans; 21,22 compared with other model organisms zebrash larvae are transparent, their internal structure and organ development process can be observed directly through the microscope, thus the transparency of zebrash embryos and larvae provides experimental advantages for studying the accumulation sites of uorescent-labeled CDs; Zebrash's blood-brain barrier is similar to that of human, which can be used as the basis for drug screening in the central nervous system; 23 Zebrash's nervous system, including central nervous system and peripheral nervous system, is similar to that of human.][29] Chlorella was prepared into nano CDs, so that, on the one hand, the sterilization and anti-inammatory effect of the CDs could be applied, while on the other hand, the size of the chlorella could be reduced to achieve the purpose of promoting absorption.However, there is no report on the application of chlorella-derived CDs to zebrash.In order to study the possible effects of chlorella-derived CDs on organisms, herein, the eggs and adult sh of zebrash were soaked with chlorella-derived CDs, and the effects of chlorella-derived CDs on zebrash were studied by transcriptome sequencing and intestinal microbial sequencing (Fig. 1).

Structural characterization of the CDs
The XRD patterns of the chlorella-derived CDs indicated the presence of graphitic carbon with a peak at approximately 24°( Fig. 2a).In the UV-vis absorption spectrum, the CDs showed a distinct peak around 280 nm (Fig. 2b).The excitationdependent uorescence of chlorella carbon was observed within an excitation wavelength range from 420 nm to 500 nm; with an increase in the excitation wavelength, there was a red-shi in the emission peak position.The CDs exhibited the strongest emission peak at 420 nm corresponding to blue uorescence.Fourier-transform infrared spectroscopy analysis revealed that carbonyl groups (C]O) and hydroxyl groups (-OH) were present in the chlorella-derived CDs, as indicated by stretching vibration characteristic peaks at 1660 cm −1 and   deconvoluted C 1s and O 1s core-level XPS spectra revealed the presence of C-C bonds and functional groups, such as C-N and C]O, in the CD domains (Fig. 3b and d).The N 1s XPS spectrum could be deconvoluted into one peak attributed to C-N (Fig. 3c).

Fluorescence imaging and histological observations of the CDs in zebrash
Zebrash exposed to CD solutions at concentrations of 0.5, 1, and 2 mg mL −1 exhibited excellent uorescence imaging results (Fig. S2 †).However, when exposed to high-concentration CD solution, a low survival rate was observed, which may be due to the high concentration of the CD solution and excessive color, which affected the circadian rhythm of the zebrash eggs, thus affecting their growth and development.Notably, the mortality rate of zebrash in the low-concentration CD solution (0.5 mg mL −1 ) showed no signicant difference compared with the control group.Therefore, this concentration was selected for further exposure of the zebrash during the uorescence imaging and transcriptome experiments.The uorescence intensity of the zebrash eggs did not increase with prolonged exposure until reaching 96 hpf; instead, CDs accumulated within the yolk sac aer hatching (Fig. S2 †).To investigate the metabolic time of CDs in the zebrash eggs, a blank solution was used as a replacement for the CD solution aer soaking for 48 hpf for observation purposes.Aer discontinuing exposure for 48 hpf, there was a signicant decrease in uorescence intensity within the fry, indicating that the water-soluble CDs could not accumulate within the organisms (Fig. 4).
Aer soaking the zebrash in CD solution (1 mg mL −1 ) for 12 h, it was observed that the CDs could diffuse into the sh through the eyes, back, and tail of the selected sh (Fig. 5).The control zebrash developed severe enteritis, intestinal villi rupture, and cellular vacuolation; however, the cellular vacuolation and intestinal villi rupture decreased aer immersion in CD solution (1 mg mL −1 ) for 12 h (Fig. 6).It is speculated that inammatory lesions, such as intestinal wall rupture, gradually decreased in zebrash under the inuence of the CDs.

Transcriptomic analysis of zebrash
High-throughput sequencing was employed for the transcriptome sequencing analysis of the zebrash eggs exposed for 48 hpf (experimental group named CDs-S) and 96 hpf (experimental group named CDs-L).The sequencing results revealed the upregulation of more genes in the zebrash eggs under short-term exposure to CD solution.With the prolonged exposure time, differential gene expression increased and became predominantly upregulated (Fig. S3 †).The differentially expressed genes induced by CD exposure in zebrash eggs were primarily associated with metabolismrelated pathways.Lipid metabolism and amino acid metabolism may be affected by exposure to the CD solution in these eggs.In addition, some genes were concentrated within genetic information processing-related pathways, suggesting a potential impact on genetic information processing in zebrash eggs.Both GO and KEGG databases were used to select the 10 pathway items with the most signicant enrichment to be displayed in the gure.If there were fewer than 10 enriched pathway items, all of them were displayed (Fig. S4 and S5 †).The CDs-S differential genes were predominantly enriched in 651 GO functional categories, with a particular concentration in the extracellular region, extracellular space, and formation of the primary germ layer.The CDs-L differential genes exhibited enrichment across 817 GO functional categories, primarily within organelles and intracellular compartments.The CDs-S group was mainly enriched in endocytosis (Fig. S4c †).CDs-L differential genes were signicantly enriched in four pathways: spliceosome, protein processing in the endoplasmic reticulum, RNA transport, and ribosome biogenesis in eukaryotes.It is speculated that short-term exposure to CD solution may impact protoderm formation in eggs, while prolonged exposure could induce changes in egg organelles (Fig. S4d †).
During the experiment, it was found that CDs may affect the Wnt signaling pathway of sh eggs.Most of the genes in the signaling pathway were down-regulated when soaked for a short time, while most of the genes were up-regulated when soaked with CDs for a long time.Wnt is associated with zebrash embryonic development, 30 and Wnt/b-catenin signaling is essential for early sh swim bladder development, 31 while Wnt may also affect zebrash brain development. 32dult zebrash exposed to CDs (1 mg mL −1 ) for 12 h were selected for transcriptome sequencing and intestinal ora sequencing of the brain (referred to as CDs-B) and intestine (referred to as CDs-I).In response to CD exposure, zebrash brain tissue exhibited fewer differentially expressed genes, which were mostly down-regulated and enriched in metabolic pathways related to dopamine secretion, indicating a potential decrease in dopamine levels (Fig. S5a and b †) when exposed to >200 mg mL −1 CD solution.The CDs-B group showed enrichment in 112 GO functional categories, specically concentrated in the extracellular region and cell cycle.The CDs-I group displayed enrichment across 130 GO functional categories, particularly those associated with immune system processes and the extracellular region.The differential genes were enriched, and the KEGG enrichment point diagram of the differential genes graphically displays the KEGG enrichment analysis results.
The differential genes of the CDs-B group are enriched in 11 metabolic pathways, most of which are enriched in MAPK signaling pathway, dopaminergic synapse.The CDs-B differential genes were enriched in one metabolic pathway, and were enriched in the PSteroid hormone biosynthesis in the endoplasmic reticulum (Fig. S5c †).The CDs-I group differential genes were enriched in 7 metabolic pathways, and they were also enriched in cytokine-cytokine receptor interaction, protein digestion and absorption, glycerophospholipid metabolism, etc (Fig. S5d †).
Compared with the control group, the CDs affected the intestinal ora abundance of zebrash.The highest abundance was proteobacteria (92.86%), and the highest abundance of CDs-I group was proteobacteria (65.33%) and rmicutes (15.89%) (Fig. S6 †).As can be seen from the results of a diversity analysis, the Chao1, Shannon, and Simpson indexes all showed an increase in intestinal ora diversity in the CDs-I group.The inuence of CDs on the intestinal ora of zebrash was mainly reected in the diversity of intestinal ora.Aer soaking in CDs, the diversity of the intestinal ora of zebrash increased, while the abundance of proteobacteria in the intestinal ora decreased, while the abundance of fusobacteria increased.

Conclusions
The uorescence imaging results of zebrash demonstrated that chlorella-derived CDs exhibited a pronounced uorescence imaging effect.Within a specic concentration range, the CDs did not signicantly impact the survival data of zebrash adults and eggs.However, at the molecular level, the CDs inuenced the brain, intestine, and eggs.It was observed that the carbon dots (CDs) exhibited excellent uorescence imaging capabilities in zebrash, indicating their favorable biocompatibility.Transcriptomics and 16S gene analysis were employed to investigate the molecular-level effects of the CDs on zebrash embryo development, brain tissue function, and intestinal tissue health.The ndings revealed a signicant impact of CDs on the dopaminergic pathway in the zebrash brain.By up-regulating the D2 receptor within this pathway, the CDs induced K + channel opening, resulting in heightened sh activity levels and enhanced external performance.The transcriptome sequencing of zebrash intestinal tissues demonstrated that the CDs promoted complement coagulation cascade activation within the intestine, suggesting potential anti-inammatory effects.Furthermore, the analysis of the intestinal ora indicated that the CDs increased diversity and improved the overall environment of gut microbiota.This study aimed to explore chronic toxicity and genotoxicity associated with the CDs in zebrash while providing valuable insights for their application in biology and medicine.Through the analysis of the zebrash gut microbiota sequencing data, it was observed that the CDs could augment the diversity of intestinal ora and establish a new equilibrium.The affected pathway of the CDs may inspire the development of chlorella-derived CDs as gene-related inhibitors or promoters, thereby offering new perspectives for future nanodrug applications.

Fig. 1
Fig. 1 Schematic illustration of the fluorescence imaging and toxicology study of chlorella-derived CDs in zebrafish.

Fig. 2
Fig. 2 (a) XRD patterns of the CDs; (b) UV-visible absorption spectrum of the CDs; (c) FTIR spectrum of the CDs; (d) fluorescence spectra of the CDs.

Fig. 3
Fig. 3 (a) XPS survey spectrum of the CDs and deconvoluted C 1s (b), N 1s (c), and O 1s (d) spectra of the CDs.